Despite years of effort, a licensed malaria vaccine is not available. One of the obstacles facing the development of a malaria vaccine is the extensive heterogeneity of many of the malaria vaccine antigens. Potential vaccine antigens that have been evaluated in people thus far have not elicited a protective immune response.
Malaria kills approximately 863,000 people every year. Although a variety of anti-malarial drugs exist, the cost of these drugs can be prohibitive in the relatively poor areas of the world where malaria is endemic. The widespread use of the most commonly employed drugs has also resulted in the expansion of drug-resistant parasites, rendering many of these drugs ineffective. In the absence of inexpensive, highly potent drugs, vaccination represents the most cost-effective way of supplementing traditional malaria interventions.
A successful malaria vaccine will need to protect people against a large population of antigenically diverse malaria parasites. A vaccine based on a single isolate of a single antigen may not be able to elicit an immune response that is broad enough to protect individuals against this heterogeneous population. One way to potentially enhance the efficacy of antigen-based vaccines, or any other subunit malaria vaccine, would be to incorporate additional malaria antigens into the vaccine, thereby broadening the immune response elicited by the vaccine.
Malaria vaccine development efforts have focused almost exclusively on a handful of well-characterized Plasmodium falciparum antigens. Despite dedicated work by many researchers on different continents spanning more than half a century, a successful malaria vaccine remains elusive. Sequencing of the P. falciparum genome has revealed more than five thousand genes, but has given no indication which of these five thousand genes will be useful, or how to identify potential vaccine targets.
Malaria is caused by mosquito-borne hematoprotozoan parasites belonging to the genus Plasmodium. Four species of Plasmodium protozoa (P. falciparum, P. vivax, P. ovale and P. malariae) are responsible for the disease in humans. Others cause disease in animals, such as P. yoelii and P. berghei. P. falciparum accounts for the majority of infections and deaths in humans. Malaria parasites have a life cycle consisting of four separate stages. Each one of these stages is able to induce specific immune responses directed against the parasite and the correspondingly occurring stage-specific antigens, yet naturally induced malaria does not protect against reinfection.
Malaria parasites are transmitted to mammals by several species of female Anopheles mosquitoes. Infected mosquitoes deposit the sporozoite form of the malaria parasite into the mammalian skin during a blood meal, which subsequently invades the bloodstream. Sporozoites remain for a few minutes in the circulation before invading hepatocytes. At this stage, the parasite is located in the extra-cellular environment and is exposed to antibody attack, mainly directed to the circumsporozoite (CS) protein, a major component of the sporozoite surface. Once sporozoites invade hepatocytes, the parasite differentiates, replicates and develops into a schizont. During this stage, the invading parasite will undergo asexual multiplication, producing up to 20,000 daughter merozoites per infected hepatocyte cell. During this intracellular stage of the parasite, the host immune response includes T lymphocytes, especially CD8+ T lymphocytes. After 10-14 days of liver infection, thousands of newly formed merozoites are released into the bloodstream and invade red blood cells (RBCs), becoming targets of antibody-mediated immune response and T-cell secreted cytokines. After invading the erythrocytes, the merozoites undergo several stages of replication, transforming into trophozoites, and schizonts, which rupture to produce a new generation of merozoites that subsequently infect new RBCs. This phase (erythrocytic) of the parasite stimulates a strong humoral response that can block merozoite invasion of RBCs and usually confers protection against pathology associated with this phase. The erythrocytic stage is associated with overt clinical disease. A smaller number of trophozoites may develop into male or female gametocytes, which are the parasite's sexual stage. When susceptible mosquitoes ingest gametocytes, the fertilization of these gametes leads to zygote formation and subsequent transformation into ookinetes, then into oocysts, and finally into sporozoites, which migrate to the salivary gland to complete the cycle.
The two major arms of the pathogen-specific immune response that occur upon entry of the parasite into the body are cellular and humoral. The one arm, the cellular response, relates to CD8+ and CD4+ T cells that participate in the immune response. Cytotoxic T lymphocytes (CTLs) are able to specifically kill infected cells that express pathogenic antigens on their surface. CD4+ T cells or T helper cells support the development of CTLs, produce various cytokines, and also help induce B cells to divide and produce antibodies specific for the antigens. During the humoral response, B cells specific for a particular antigen become activated, replicate, differentiate and produce antigen-specific antibodies.
Both arms of the immune response are relevant for protection against a malarial infection. When infectious sporozoites travel to the liver and enter the hepatocytes, the sporozoites become intracellular pathogens, spending little time outside the infected cells. At this stage, CD8+ T cells and CD4+ T cells are especially important because these T cells and their cytokine products, such as interferon-γ (IFN-γ), contribute to the killing of infected host hepatocytes. Elimination of the intracellular liver parasites in the murine malaria model is found to be dependent upon CD8+ T cell responses directed against peptides expressed by liver stage parasites. Depletion of CD8+ T cells abrogates protection against sporozoite challenge, and adoptive transfer of CD8+ T cells to naïve animals confers protection.
When a malarial infection reaches the erythrocytic stage in which merozoites replicate in RBCs, the merozoites are also found circulating freely in the bloodstream for a brief period until they invade new erythrocytes. Because the erythrocyte does not express either Class I or II MHC molecules required for cognate interaction with T cells, it is thought that antibody responses against the parasite are most relevant at the blood stage of the parasite lifecycle. In conclusion, a possible malaria vaccine approach would be most beneficial if it would induce a strong cellular immune response as well as a strong humoral immune response to tackle the different stages in which the parasite occurs in the human body.
Current approaches to malaria vaccine development can be classified according to the different developmental stages of the parasite, as described above. Three types of possible vaccines can be distinguished. The first is pre-erythrocytic vaccines, which are directed against sporozoites and/or schizont-infected hepatocytes. Historically, this approach has been dominated by (CSP)-based strategies. Since the pre-erythrocytic phase of infection is asymptomatic, the goal of a pre-erythrocytic vaccine would be to confer sterile immunity, mediated by humoral and cellular immune response, and thereby prevent latent malaria infection. This goal has not been met by any known treatment.
The second type of vaccine approach is asexual blood stage vaccines, which are directed against either the infected RBC or the merozoite itself, are designed to minimize clinical severity or prevent infection if antibodies prevent merozoites invading erythroctyes. Attempts to create such vaccines so far have failed to sufficiently reduce morbidity and mortality or prevent the parasite from entering and/or developing in the erythrocytes. Transmission-blocking vaccines are designed to hamper the parasite development in the mosquito host. Attempts to create this type of vaccine so far have failed to reduce population-wide malaria infection rates.
The final type of vaccine approach is combination malaria vaccines that target multiple stages of the parasite life cycle. This approach attempts to develop multi-component and/or multi-stage vaccines. Attempts to create such vaccines so far have failed to effect sufficient protection. As a result of these failures, there is currently no commercially available vaccine against malaria.
Immunization of rodents, non-human primates, and humans with radiation-attenuated sporozoites (RAS) has been found to confer protection against a subsequent challenge with viable sporozoites. However, the expense and the lack of a feasible large-scale culture system for the production of irradiated sporozoites, the relative short-term efficacy, lack of cross-strain protection, and the need to be delivered intravenously have been obstacles to the development of such vaccines.
The CS protein is the only P. falciparum antigen demonstrated to prevent malaria infection when used as the basis of active immunization in humans against mosquito-borne infection. The protection levels for this antigen, however, are not high enough to support a viable therapy. In theory, vaccine protection levels should be above 85% in order to be a viable therapy. With protection lower than that, mutants that are more virulent may escape in endemic areas. CS antigen-based vaccines have demonstrated an efficiency of only about 50% and that protection does not last more than a year. Nevertheless, this is still the best known antigen response prior to the present disclosure.
The entire genomic sequence of P. falciparum has been sequenced. See Bowman et al., Nature, 400: 532-538 (1999); Gardner, et al., Nature, 419: 498-511 (2002). Another human malaria parasite, P. vivax, has also been sequenced. See Carlton et al., Nature, 455: 757-763 (2008). The rodent malaria parasite, P. yoelii has also been sequenced. See Carlton et al., Nature, 419: 512-519 (2002). Despite this, however, the development of efficacious anti-malaria vaccines has been severely hampered by the inability to identify promising antigens. Sequencing of the P. falciparum, P. vivax, and P. yoelii genomes has resulted in the identification of 5,369, 5,433, and 5,675 genes, respectively. Knowledge of these sequences alone, however, will not likely result in new vaccine constructs. Consequently, only 0.2% of the P. falciparum proteome is undergoing clinical testing, and these tests have failed to induce high grade protection in volunteers.